need help on RNA isolation from plant

Sharon Regan sregan at pnfi.forestry.ca
Thu Mar 30 13:18:17 EST 1995


A few weeks ago I asked a similar question on the Arabidopsis net, this is a 
summary of the replies.

Good Luck
Sharon Regan
Petawawa National Forestry Institute
Chalk River Ontario K0J 1J0
email: sregan at pnfi.forestry.ca



Procedure #1 from Linda Deverno

Isolation of DNA from small amounts of plant tissue from Doyle and Doyle, BRL
Focus 12: 13-15, 1990, modified in Forest Biotechnology lab at NCSU.

1.  Make up 700uL fresh 2X CTAB buffer (add proteinase K and mercaptoethanol)
for each sample.  

2.  Grind tissue thoroughly in small amount (25-50uL) of buffer using
disposable pestle and motorized stirrer.  Add  3 volumes more of 2X CTAB
buffer.  Place at 65C for 30 minutes.

3.  Add equal volume chloroform:isoamyl alcohol (24:1) to each tube.  Vortex
briefly and gently.  Spin 5 minutes in microfuge.

4.  Remove aqueous (top) layer to new tube.

5.  Optional extra cleaning step.  Add 1/10 volume 10%CTAB (in 0.7M NaCl),
vortex gently.  Repeat steps 4 and 5.

6.  Add equal volume isopropanol (-20C) or 2.5 volumes ethanol.  Invert tube
several times and let tube sit at room temperature for 30 minutes.

7.  Spin 15 minutes in microfuge, pour off supernatant.  Rinse with 70%
ethanol, spin 15 minutes.  Remove ethanol, dry precipitate.

8.  Dissolve DNA in 25uL TE (10/1mM)

2X CTAB Extraction Buffer	(for 100mL)

2% CTAB				2g
1.4M NaCl				8.12g
20mM EDTA				4mL of 0.5M
100mM Tris Hcl, pH 8.0		10mL of 1M
2% polyvinylpyrollidone		2g
water (HPLC)				to 100mL
Note:  CTAB will only go into solution after the salt is added.
Add upon use:		0.2% 2-mercaptoethanol		2uL/mL buffer
			10mg/mL (stock) proteinase K	0.5mg/mL buffer


 Procedure #2 from Joel Kreps

1. Grind tissue in liq. Nitrogen, transfer sand-like grindate to a 1.5 ml
tube, put a maximum of 0.5 ml worth of tissue per tube.

2. Add 550 ul of Extraction buffer, vortex 20 sec. put tube on ice until all
the preps are done.

3. Add 550 ul of h2O sat. phenol, vortex 20 sec, spin 5 min at top speed.

4. Remove aqueous layer, re-extract it with phenol.

5. Extract aqueous layer with chloroform, sometimes I have tissue with a lot
of polysachharide and I will do up to 3 chlorof. extractions.

6. EtOH ppt: 1/10 vol 3 M NaAcetate, pH 5.0 (or 5.2), 2 vol of 95% EtOH (100%
is okay). -70C for 30 min to overnight.  Pellet, 10 min in microfuge at full
speed.  Completely remove supernatant, do not bother drying the pellet.

7. Wash the pellet (by pipetting up and down) with 300 ul 2M LiCl.  Allow the
pellet to sit in the LiCl for 30 min to overnight on wet ice (or 4C).  Pellet,
10 min.  Completely remove supernatant, do not dry pellet, resuspend in 400 ul
h2O.

8. EtOH ppt as in step 6.  Wash the pellet with 70% etOH.  Dry the pellet and
resuspend in H2O.  If I use a full .5 ml of grindate, I add 50 ul and get 1 to
4 ug/ul.

Extraction buffer

0.2 M Tris, pH 9.0
0.4 M LiCl
25 mM EDTA
1% SDS


 Procedure #3

Verwoerd, TC, Dekker, BMM, Hoekema, A (1989). A small-scale procedure 
for the rapid isolation of plant RNAs.  NAR 17: 2362
Modified by Terry Delaney (Ciga Geigy)
Adapted to Malmberg lab
This methods works well for small amounts of Arabidopsis tissue.  3-4 large leaves or up to quarter sized plant is maximum amount of tissue that works well.   Larger amounts of tissue result in poor quality RNA.

Materials:
Extraction buffer: 	100 mM LiCl
			100 mM Tris pH 8.0
			 10 mM  EDTA
			  1 %  SDS
Phenol
Chloroform
3M NaAcetate
Ethanol
Liquid Nitrogen
80oC heating block or water bath
Pellet Pestles for 1.5 ml microcentrifuge tubes
Loading mix: 		per sample
			12.5 ul formamide
			 4.25 ul formaldehyde
			  2.5 ul MES buffer
			  0.5 ul ethidium bromide (10mg/ml)
			  0.5 ul BPB loading dye

Before starting make sure the heating block is set at or just below 80oC.   CAUTION phenol will violently boil from a test tube upon heating above 80oC causing hot phenol to spray all over you and your lab bench!!! 
1.  Mix extraction buffer with and equal volume of phenol (1:1) and place in heating block
2.  Label three microcentrifuge tubes for each sample you are processing
3.  Freeze a microfuge tube in liquid nitrogen, then fill the tube with liquid nitrogen and place in a tube rack.
4.  Quick freeze your plant material by plunging it into the tube full of liquid nitrogen.  
5.  Freeze the pestle and grind the tissue to a fine powder. 
6.  Add 500 ul of the hot extraction buffer/phenol mix to the frozen tissue and thaw by vortexing.
7.  Add 250 ul of chloroform and vortex
8.  Spin 5 min in microfuge to separate the phases
9.  Remove the aqueous phase (top) to a clean tube
10.  precipitate the nucleic acids by adding 1/10 vol. of  3M Sodium Acetate and 2 volumes of ethanol mix incubate -20oC 20 min.
11.  A lithium chloride ppt step can be added here to remove contaminating DNA from the sample if this will pose a problem in later experiments. (not usually needed for Northern blotting).  
12.  Resuspend RNA in 5.5 ul of water and add 19.5 ul of loading mix.  
13.  Denature samples and load onto gel 


 Procedure #4  from Margaret Hollingsworth:

We have a set RNA isolation procedure that works really 
well for us, especially for northern work.

1)	Grind up your sample in liquid nitrogen

2)	Add 50 mM NaAc pH5.5 1%SDS 1 mM ATA
	add around a ml per 0.5 g of powdered tissue
	ATA is aurin tricarboxylic acid - it's an RNase inhibitor that
	you can buy cheaply from Sigma.  The concentration isn't 
	critical - from 1 to 10 or more mM works well

3)	phenol/chloroform extract SIX times
	You'll lose some RNA here, but if it's not clean, it'll hang
	in the well.  If you're worried about yield, you can always
	back-extract the p/c.

4)	Ethanol precipitate.  The pellet will be reddish, since the
	ATA will also precipitate.  ATA runs faster than bromophenol
	blue on a gel, so it will separate from the RNA at that point.

A side note - ATA inhibits all kinds of nucleic acid interacting enzymes.
So if you use this prep for anything other than a northern (translation,
primer extension, etc), you'll need to get rid of the ATA - run it over
a spin column (G50 works fine).  ATA will be held back in the beads.


 Procedure #5 Carl W. Garnaat recommends: 

I recommend the Pharmacia QuickPrep micro RNA purification kit. I have
successfuly used this with as little as two maize spiklets. I loaded 10% of
the product on a lane for Northerns and very easily detected anther specific
transcripts.  The quality of the RNA was very good. Each spikelet has two
florets, only one with three anthers at the right developmental stage. The
transcripts were relatively abundant.  The yield with this kit essentially
maxed out with 8 spikelets.

This kit is very easy and the mRNA is directly isolated from up to 100 mg plant
material within one hour.  One thing I really like is that Pharmacia quality
controls this kit with Mung bean since plant tissues especially mung bean
have substantially more RNase than blood cells.

I froze the tissues first in liquid N in a Kontes tube, added the extraction
buffer and used the Kontes pestle with a drill to homogenize the tissue, (the
buffer thaws during this step). 






Procedure #6 from Kevin Seeley 
Seeley et al., 1991  Plant Cell  4, 29-38


 Procedure #7 from Brenda Shirley

RNA Prep from Seedlings
(mainly from Altenbach and Howell, 1981)
This works well for Arabidopsis seedlings (off plates), 3 week-old plants,
flowers, whatever.

1. Harvest tissue (1000 3 day old seedlings, for example, but I've used
less tissue with the same volumes or more tissue and scaled up, both with
good results) into liquid nitrogen and store at -70 C.

2. Grind tissue in liquid nitrogen using a pre-cooled mortar and pestle.

3. Transfer to a 15 ml Falcon tube containing 1.5 ml water-saturated phenol
and 1.5 ml RNA extraction buffer:

0.2 M Tris, pH 9        4 ml 1M
0.4 M LiCl              4 ml 2 M
25 mM EDTA              1 ml 0.5M
1% SDS                  1 ml 20%
                        10 ml H2O

4. Vortex well.  Spin 5 min, 3K rpm.

5. Remove aqueous phase and re-extract with 1.5 ml phenol.

6. Extract 2X with 1.5 ml chloroform.

7. Transfer aqueous phase to a sterile plastic culture tube (e.g. Fisher's
14-956-1J).  Precipitate with 1/10 vol (~150 ul) 3M NaOAc, pH 5 and 2
volumes (~3 ml) EtOH.  Place at -20 C, 1 hour.

8. Spin at 8K, 15 min.

9. Resuspend pellet in 0.3 ml 2M LiCl and transfer to a microfuge tube.
Vortex well.  Spin in microfuge for 5 min at 4 C.

10.  Resuspend pellet in 0.3 ml H2O.  Vortex well.  Add 30 ul 3M NaOAc, pH5
and 600 ul EtOH.  Place at -20 C, 1 hour.

11. Pellet RNA, wash 2X with 80% EtOH.

12. Resuspend pellet in 50 ul H2O.  Use 5 ul in 750 ul to determine OD.
Store remainder at -80 C.

Works quickly (probably part of why it works so well) and easily and the
RNA seems to remain in good shape for a long time.  The only solutions you
really need to DEPC-treat are the LiCl, NaOAc, and water (also used to make
80% EtOH).






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